The PhosphoProtein Purification Kit, which is based on affinity chromatography, delivers a complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. In addition, the ratio of phosphorylated to unphosphorylated forms of proteins can easily be determined. The drastic reduction of the complexity of each fraction is especially useful when studying proteins of low abundance. The PhosphoProtein Purification Kit includes columns, reagents, buffers, and Nanosep Ultrafiltration Columns for efficient concentration and desalting of protein fractions.

The PhosphoProtein Purification Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.

Highly specific separation of phosphorylated proteins.

Non-stimulated Jurkat cells were radioactively labeled in vivo using 32P. The cell lysate was processed using the PhosphoProtein Purification Kit and the radioactivity in each fraction measured. (Data kindly provided by Gudrun Rehg and Sascha Dammeier, Byk Gulden, Konstanz, Germany.)

Phosphoprotein purification procedure.

Complete separation of unphosphorylated and phosphorylated proteins.

Complete Separation of Unphosphorylated and Phosphorylated Proteins. Protein-specific immunodetection of [A] unphosphorylated HSP-60 protein, and [B] phosphorylated p44 and p42 mitogen-activated protein kinase (MAPK) proteins. F: flow-through; E: eluate fractions. The antibody used to detect MAPK recognizes an epitope containing phosphorylated residues at Thr202 and Tyr204 in the p44 (upper band) and p42 (lower band) MAPK amino acid sequences. The absence of unphosphorylated HSP-60 in the eluate fraction and the absence of phosphorylated MAPK in the flow-through fraction demonstrate the complete separation of phosphorylated proteins using the PhosphoProtein Purification Kit.

Efficient enrichment for 2D-PAGE phosphoproteome analysis

HeLa cells were treated with the phosphatase inhibitors Calyculin (100 nM) and orthovanadate (1 mM) for 20 min at 37°C. [A] After harvest and cell lysis, an aliquot of the lysate was separated by 2D-PAGE and proteins visualized using a non-specific total protein stain (blue) and a phosphoprotein-specific stain (orange, images overlaid). [B] A second aliquot was processed using the PhosphoProtein Purification Kit and the eluate (containing phosphoproteins) treated as before. Overlapping protein spots are colored black. Image analysis indicated that 87% of eluted proteins are phosphorylated.

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SDS-PAGE analysis of flow-through and eluate fractions.

Flow-through fractions (left-hand lanes) and eluates (right-hand lanes) from cell lysates processed using the PhosphoProtein Purification Kit. Both fractions were concentrated by a factor of 10 before loading onto the gel. Proteins were visualized by Coomassie staining.

The PhosphoProtein Purification Kit is based on an affinity chromatography process and provides complete separation of phosphorylated and unphosphorylated proteins from a cell lysate, and therefore facilitates investigation of the phosphorylation status of both entire cells and specific proteins. Phosphorylated proteins can be identified after blotting using highly specific mouse monoclonal PhosphoThreonine and PhosphoSerine Antibodies, which recognize an epitope consisting of either a phosphothreonine or phosphoserine residue, irrespective of the surrounding amino acids. See figure Complete separation of unphosphorylated and phosphorylated proteins.

Procedure

Each PhosphoProtein Purification Column can be used to purify phosphorylated proteins from 107 eukaryotic cells (equivalent to approximately 2.5 mg total protein). Cells are lysed in the supplied lysis buffer, which contains a detergent and nucleases to resolve protein complexes, and protease inhibitors to prevent protein degradation. Cleared lysates are loaded onto the PhosphoProtein Purification Column, where phosphorylated proteins in the lysate bind to the column, while unphosphorylated proteins are found in the flow-through fraction. After a wash step, phosphorylated proteins are eluted from the column (see flowchart Phosphoprotein purification procedure ). Typically approximately 10% of the total protein loaded is in the phosphorylated fraction (depending on cell type and status). Both fractions retain biological activity and can be further purified if desired. Nanosep Ultrafiltration Columns are supplied with the PhosphoProtein Purification Kit to enable efficient concentration and desalting of protein fractions. The table shows typical protein yields in each fraction after separation using the PhosphoProtein Purification Kit.

Yields of phosphorylated proteins obtained using the PhosphoProtein Purification Kit.

Cell type

Number of cells processed

Total protein in cell lysate (µg)

Protein loaded onto column (µg)

Protein in eluate (µg)

Percentage phosphorylated proteins

CHO

1.5 x 107

3400

2500

300

12%

NIH 3T3

n.d.

2750

2500

165

7%

293

1.5 x 107

3650

2500

200

8%

Cos-7

4.5 x 106

1700

1700

120

7%

Huh-7

8.5 x 106

2650

2500

235

9%

HT 29

n.d.

n.d.

2500

200

8%

LT 23

n.d.

n.d.

2500

275

11%

HeLa S3

1.8 x 107

5950

2500

280

11%

HeLa Acc57

6.6 x 106

2500

2500

235

9%

Applications

The PhosphoProtein Purification Kit provides complete separation of phosphorylated and unphosphorylated protein fractions for research in: